Antimicrobial resistance for both Gram-positive and -negative pathogens has spread worldwide at an increasing rate, presenting a challenging global health problem. Even in the U.S., hospital- and community- acquired methicillin-resistant Staphylococcus aureus (MRSA) have became more virulent and can cause a greater spectrum of illness than their predecessors. In particular, MRSA has been found to exhibit an alarming ability to strike otherwise healthy people. One strategic approach to preventing antibiotic resistance is to treat bacterial infections with more than one drug at a time. Synergies of interaction can both increase the efficacy of the combination and reduce the probability of the bacterium's surviving by developing a double fortuitous mutation conveying resistance. We propose to investigate the potential of amphipathic cationic cyclopeptides, which are capable of disrupting the bacterial membranes, to elevate the efficacy of some clinically used antibiotics against several types of Gram-positive and -negative bacteria. These amphipathic cationic cyclopeptides, like the better-known polycationic peptides, could enhance the uptake of small hydrophobic molecules into the bacterium and present synergistic effects. The long-term goal of this project is to design-fast acting antimicrobials that may lead to novel antimicrobial therapeutic routes, for example, they can provide synergism in combination antimicrobial therapy or act as a scaffold for antibiotic targeting. The objectives of this project are (i) to develop a clearer understanding of the interaction of cationic cyclooctapeptides with bacterial membranes and their associated divalent metal ions (Ca2+ and Mg2+) by a structure-and-binding correlation study, (ii) to identify the origins of the binding specificity between the donor atoms on the cyclooctapeptide and Ca2+ or Mg2+ by solid state studies, and (iii) to utilize these understandings to develop effective antimicrobials. In working towards these project objectives, we will synthesize a series of amphipathic cationic cyclopeptides and their bis-analogs by microwave-assisted solid-phase-peptide synthesis and evaluate their in vitro intrinsic antimicrobial activities against several strains of bacteria by conducting minimum inhibitory concentration (MIC) assays and bacterial killing assays. Their interactions with membrane-mimicking detergents and divalent metal ions associated with stabilizing bacterial membrane will be studied by isothermal titration calorimetry (ITC), circular dichroism and X-ray diffraction methods. We will also investigate their interactions with some common clinically used antibiotics and assess for synergistic, additive, or antagonistic effects using minimum inhibitory concentration (MIC) assays and checkerboard titrations. These fundamental studies will advance our understanding of the mechanism of action of these antimicrobial cyclopeptides with bacterial membranes and their associated metal ions, and demonstrate their potential in enhancing the efficacy of some antibiotics and in prevailing over antibiotic resistance. Consequently, these research findings could be useful in improving public health. PUBLIC HEALTH RELEVANCE: Antimicrobial resistance for both Gram-positive and -negative pathogens has spread worldwide at an increasing rate, presenting a challenging global health problem. The goal of this project is to design fast-acting antimicrobials that may lead to novel antimicrobial therapeutic routes. These fundamental studies are expected to demonstrate the potential of bacterial membrane-disrupting molecules in enhancing the efficacy of some antibiotics and in prevailing over antibiotic resistance.